Nanosecond pulsed electric field inhibits malignant melanoma growth by inducing the change of systemic immunity

Abstract

Background: Nanosecond pulsed electric fields (nsPEFs) showed an inhibitory effect on proliferation of malignant melanoma. In this study, the growth of melanoma were inhibited by changing the systemic immunity .

Material and methods: C57BL/6 mice with B16 malignant were exposed to 200 pulses of 100 ns duration, 30kV/cm. The mice were executed four days later. T lymphocyte has been extracted from spleen. Cell viability was evaluated by CCK-8 assay. CD3+CD4+ T cells, CD3+CD8+ T cells, regulatory T cells (Treg) and myeloid-derived suppressor cells (MDSC) were analyzed by flow cytometry . TNF-α, IL-2, IL-10, TGF-β, IFN- γ levels in supernatants were assessed by ELISA.

Results: C57 malignant melanoma model were established successfully. After the treatment of nsPEFs(30 kV/cm 100 ns 200p), the numbers of T lymphocytes were increased.CD3+ CD4+ T cells changed from 48% to 51.2%;CD3+CD8+T lymphocytes increased from 39.6% to 40.4%.Treg cells reduced from 4.3% to 2.4%,MDSC decreased by 39.0% to 19.7% . In addition, the level of TNF-α, IL-2 were increased (P < 0.05) and the level of IL-10 were decreased (P < 0.05) and the level of TGF-β and IFN-γ remained stable (P > 0.05).

Conclusions: Tumor growth can be effectively inhibited by nsPEFs in vivo, which activate targets of immune respones, accumulation of inflammatory cells and immune cytokines.

Figure 1

The change of C57BL/6 mice with B16 melanoma cells after treatment and extracted T lymphocytes from the spleen. 7 days after inoculation B16, the distribution of subcutaneous tumor can be found. Tumor-bearing mice treated by nsPEFs (Fig. 1B). Untreated tumor (Fig. 1A). The tumor with 30 kv,100 ns,100 pulses treated (Fig. 1C); the tumor with 30 kv,100 ns, 200 pulses treated (Fig. 1D). 4 days later, extracted T lymphocytes from the spleen (Fig. 1E). T cells morphology under a microscope(10x) (Fig. 1F). After treatment the survival rate of T lymphocytes increased in 24 hours and 48 hours compared with the untreated group (p< 0.05) (Fig. 1G,H).

Figure 2

The flow cytometry analysis. Compared with the untreaed group, the content of CD3+CD4+T cells and CD3+CD8+T lymphocytes were increased (Fig. 2A). In contrast, the cells of treg and MDSC were reduced (Fig. 2B,C) (n≥3 independent experiments).

Figure 3

The results showed that after treatment IL-2 and TNF-α concentration increased (p< 0.05) (A,B); the level of IL-10 decreased (P< 0.05) (C). IFN-γ, TGF-β had a rising trend, but no statistical difference (P>0.05) (D,E). All data are presented as means± standard deviations (n≥3 independent experiments).